This competitive revision application is in response to NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. Heparan sulfate (HS) plays essential roles in development of various tissues. To understand how HS functions during skeletal development is of a great clinical importance. Hereditary multiple exostoses (HME) is a genetic disorder characterized by the formation of multiple osteochondromas ("exostoses"), which affects several thousands of people in the US. Individuals with HME carry heterozygous mutations of EXT1 or EXT2, which jointly encode a glycosyltransferase essential for the biosynthesis of HS. Despite the identification of its causative genes, there are a number of enigmas and unanswered questions on the pathogenic mechanism of HME. One of the most puzzling questions is the stark discrepancy between human HME manifestations and the phenotype of mice carrying the same genotype. Most importantly, both Ext1 and Ext2 heterozygous mice, which are supposed to mimic human HME faithfully, are resistant to the development of exostoses, especially in long bones. We have recently made a surprising observation that our new Ext1 conditional knockout mice (Col2a1- CreERT;Ext1flox/flox), which is based on stochastic homozygous Ext1 deletion in a small fraction of chondrocytes, develop exostoses in long bones at a 100% penetrance and phenocopy other skeletal defects of human HME to a degree not seen in any of the previous mutant mice. This competitive revision proposes to add the fourth aim (Investigate the genetic and cellular mechanisms of HME using novel HME mouse models) to characterize these mutant mice. By these studies, we wish to establish the Col2a1-CreERT;Ext1flox/flox mouse as the first mouse model directly relevant to HME, determine the significance of loss-of-heterozygocity in the pathogenesis of HME, and obtain baseline information for the future study to elucidate the signaling defects underlying osteochondroma development. PUBLIC HEALTH RELEVANCE: Heparan sulfate is essential for normal bone development, as illustrated by the existence of the human genetic bone disorder hereditary multiple exostosis (HME), which is caused by mutations of genes essential for heparan sulfate biosynthesis. In this competitive revision, we will characterize our novel conditional knockout mouse model that exhibits unprecedented levels of phenotypic similarities with human HME. The proposed studies will establish a novel mouse model for HME and generate new insights into the pathogenic mechanism of this human disorder.